Method of treating cancer cells to create a modified cancer cell that provokes an immunogenic response

ABSTRACT

The present invention relates to a delipidation method employing a solvent system useful for extracting lipids from cancer cells, thereby creating a modified cancer cell particle. Upon delipidation of the cancer cells, a portion of the cancer cell antigens remain intact. These exposed antigens, or epitopes, foster and promote antibody production. The resulting modified cancer cell particle, or portions of the cancer cell, initiate a positive immunogenic response when administered to an animal or human and help to treat, prevent or delay the onset of cancer. The present invention provides autologous and heterologous vaccine compositions comprising the modified cancer cell with a pharmaceutically acceptable carrier. The present invention provides method of administering these vaccines to treat, prevent or delay the onset of cancer.

PRIOR RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.provisional patent application Ser. No. 60/702,691 filed Jul. 27, 2005.

FIELD OF THE INVENTION

The present invention provides a delipidation method employing a solventsystem useful for extracting lipids from cancer cells, thereby creatinga modified cancer cell particle. Upon delipidation of the cancer cells,some of the cancer cell antigens remain intact. These exposed antigens,or epitopes, foster and promote antibody production. The resultingmodified cancer cell with reduced lipid content, or portions of thecancer cell, initiate a positive immunogenic response when administeredto an animal or human and help to treat, prevent or delay the onset orprogression of cancer. The present invention provides autologous andheterologous vaccine compositions comprising the modified cancer cellwith a pharmaceutically acceptable carrier. The present inventionprovides a method of administering these vaccines to treat, prevent ordelay the onset or progression of cancer.

BACKGROUND OF THE INVENTION Introduction

Cancers, of varied etiology, affect billions of animals and humans eachyear and inflict an enormous economic burden on society. Cancers can bedefined as an abnormal lump, mass of tissue, or cancerous cellsgenerated from excessive cell division, which is either benign ormalignant. Cancers include all those cancers known to physicians ofordinary skill in the medical arts, particularly physicians of skill inoncology. Cancers include, but are not limited to, those arising fromectodermal, mesodermal and endodermal cells and include cancers of theimmune system, the endocrine system, the central nervous system, therespiratory system, the reproductive system, the gastrointestinalsystem, and the integument. Such cancers include those generated byAIDS-related cancers, adrenocortical cancer, anal cancer, bladdercancer, bowel cancer, brain and central nervous system cancers, breastcancer, carcinoid cancers, cervical cancer, chondrosarcoma,choriocarcinoma, colorectal cancer, endocrine cancers, endometrialcancer, Ewing's sarcoma, eye cancer, gastric cancer, gastrointestinalcancer, genitourinary cancers, glioma, gynecological cancer, head andneck cancer, hepatocellular cancer, Hodgkin's disease, hypopharyngealcancer, islet cell cancer, Kaposi's sarcoma, kidney cancer, laryngealcancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, basalcell carcinoma, mesothelioma, myeloma, nasopharyngeal cancer,neuroblastoma, non-Hodgkin's lymphoma, esophagael cancer, osteosarcoma,ovarian cancer, pancreatic cancer, pituitary cancer, renal cellcarcinoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, sarcoma,skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer,thymus cancer, thyroid cancer, transitional cell cancer, trophoblasticcancer, uterine cancer, vaginal cancer, Waldenstrom's macroglobulinemia,Wilm's cancer, among other tumors and forms of cancer.

As with normal cells, cancer cells contain lipid as a major component ofthe plasma membrane that surrounds them. While it is not fully known whycancer cells form, in general, cancers appear to be caused by theabnormal regulation of cell division. This could be caused byabnormalities of the immune system, genetic abnormalities,radiation-caused mutations, certain viruses, sunlight, andcancer-causing agents such as tobacco, benzene, and other chemicals.When a patient is inflicted with a cancer, he incurs a number ofsymptoms, including fevers, chills, night sweats, weight loss, loss ofappetite, fatigue, malaise, shortness of breath, chest pain, diarrhea,blood in the stool or urine, among other ailments.

Eliminating cancer from a patient's body is challenging because,although cancerous cells proliferate in an uncontrolled manner, thecells do not necessarily appear to be “foreign” to the body and,therefore, are difficult to target. Existing cancer treatments tend tobe non-sufficiently targeted to the cancer cells and, therefore, arevery destructive to a patient's healthy tissue. Such treatments includeX-rays, chemotherapy, proton therapy, surgery or combinations thereof.It would be preferred if the body's immune system could be incited toexhibit a positive immune response against these cancer cells.

The human immune system is composed of various cell types thatcollectively protect the body from different foreign agents. The immunesystem provides multiple means for targeting and eliminating foreignelements, including humoral and cellular immune responses, participatingprimarily in antigen recognition and elimination. An immune response toforeign elements requires the presence of B-lymphocytes (B cells) orT-lymphocytes (T cells) in combination with antigen-presenting cells(APC), which are usually macrophages or dendrite cells. The APCs arespecialized immune cells that capture antigens. Once inside an APC,antigens are broken down into smaller fragments called epitopes—theunique markers carried by the antigen surface. These epitopes aresubsequently displayed on the surface of the APCs and are responsiblefor triggering an antibody response in defense of foreign agents.

In a humoral immune response, when an APC displaying antigens (in theform of unique epitope markers) foreign to the body are recognized, Bcells are activated, proliferate and produce antibodies. Theseantibodies specifically bind to the antigens present on the APC. Afterthe antibody attaches, the APC engulfs the entire antigen and kills it.This type of antibody immune response is primarily involved in theprevention of various infections.

In a cellular immune response, on recognizing the APC displaying aforeign antigen, the T cells are activated. There are two steps in thecellular immune response. The first step involves activation ofcytotoxic T cells (CTL) or CD8+ T killer cells that proliferate and killtarget cells that specifically represent the antigens presented by APC.The second involves helper T cells (HTL) or CD4+ T cells that regulatethe production of antibodies and the activity of CD8+ cells. The CD4+ Tcells provide growth factors to CD8+ T cells that allow them toproliferate and function efficiently.

While cancer cells are now known to express cancer-associated antigens,they are often able to evade an immune response because of their abilityto hide cancer antigens from the immune system and/or because theexposed antigens are normal, nonmutated differentiation molecules orproteins which the human immune system normally recognizes or tolerates.To effectively use immunotherapy to treat a cancer, a patient must have,or be provided with, a sufficient number of cancer-reactive lymphocytes,which can both reach the cancer site and have effector mechanisms todestroy the cancer cells.

To date, immune responses generated by cancer vaccines have been unableto overcome the escape mechanisms of cancers, including the ability totarget and infiltrate cancers, to deal with the loss of antigenicexpression by the cancer, to handle the inability of the cancer toactivate anti-cancer precursors, and to address the local presence ofimmunosuppressive factors. Some success has been observed incell-transfer therapies where autologous lymphocytes are sensitized tocancer cells ex vivo and then infused back into the patient.

One adjuvant for cancer vaccine immunotherapy uses dendritic cells (DC)that are highly potent antigen-presenting cells to provoke a positiveanti-cancer immune response in patients. Dendritic cells express MHCclass I and MHC class II molecules, co-stimulatory molecules andadhesion molecules that provide signals for the stimulation of naive Tcells, CD4+ T-helper cells, CD8+ cytotoxic T lymphocytes (CTLs), naturalkiller (NK) and thymic derived NK cells (NKT) cells. DC have thecapacity to take up various types of molecules. Consequently, DC can beloaded with tumor-associated antigens (TAAs) in various forms andadministered as vaccines.

One DC-based approach uses DC-cancer cell hybrids generated by fusion ofcancer cells with DC to combine sustained cancer antigen expression withthe antigen-presenting and immune stimulatory capabilities of DC. Inanimal models, immunization with DC-cancer cell hybrids can provide someform of anti-cancer protection or eradicate established disease. Hybridsof autologous DC comprised of cancer cell lines or primary human cancercells (including breast carcinoma cells) have been shown to induce CTLresponses against autologous cancer cell types in vitro. Recent phase Iclinical trials for the treatment of renal cell carcinoma and gliomahave demonstrated that vaccination with DC-cancer cell hybrids cansafely induce anti-cancer immune responses in patients. Traditionalfusion technology using polyethylene glycol (PEG) is hampered by a lackof reproducibility and difficulties in method standardization. As analternative, electrofusion has been used for production of DC-cancercell hybrids.” See Akporiaye, et al., “Pre-Clinical Studies of DendriticCell-Tumor Cell Fusion Vaccines to Treat Breast Cancer”.

Accordingly, what is needed is an effective delipidation process viawhich a cancer cell is modified, rather than destroyed, and invokes anautologous or heterologous immune response to prevent furtherproliferation of cancers.

What is needed is a therapeutic method and system for providing patientswith modified cancer cells capable of initiating a protective immuneresponse.

What is further needed is a way of identifying and revealingtumor-associated antigens that can be used with existing DC-cancer celltherapy techniques to provoke a positive immune response in a patient.

What is needed is a method for promoting antibody production comprisingadministering to a patient a modified cancer cell capable of initiatinga protective immune response.

SUMMARY OF THE INVENTION

The present invention solves the problems described above by providing asimple, effective and efficient method for treating cancer, preventingcancer, delaying the onset of cancer or delaying the progression ofcancer via administration of the vaccine described herein. The method ofthe present invention is effective in modifying the lipid structure of acancer cell utilizing an solvent system which does not have deleteriouseffects on the structure of cancer-associated antigens. The presentinvention employs an optimal solvent/energy system to create, viadelipidation, a modified cancer cell that has its lipid envelope atleast partially removed, thereby exposing or modifying cancer-associatedantigens that, either alone or in the form of a DC-cancer cell hybrid,can generate a positive immunologic response in a patient, providingthat patient with some degree of protection against the proliferatingcancer, preventing the occurrence or reoccurrence of the cancer, ordelaying the onset of cancer.

The present invention is also effective in producing an autologous,patient-specific vaccine against the cancer, by treating a biologicalfluid containing the cancer cell such that the cancer cell is present ina modified form. To create the vaccine, a cancer sample is removed fromthe patient (i.e. a biopsy is performed or a blood or other sample isremoved containing the cancer cells), cancer cells are isolated andpartially delipidated using an optimal solvent system which retains thestructural integrity of cancer cell antigens. In one embodiment, acancer cell, treated in this manner in order to create a modified cancercell with reduced lipid content, is administered to a recipient, such asan animal or a human, together with a pharmaceutically acceptablecarrier, and optionally an adjuvant, in order to initiate an immuneresponse in the animal or human and create antibodies that bind to theexposed epitopes of the delipidated cancer cell. In another embodiment,a cancer cell, treated in this manner in order to create a modifiedcancer cell, is for example, used to create a DC-cancer cell hybrid thatis then administered to a recipient, such as an animal or a human,together with a pharmaceutically acceptable carrier, and optionally anadjuvant, in order to initiate an immune response in the animal or humanand create antibodies that bind to the exposed epitopes of thedelipidated cancer cell.

Thus an effective method is provided, by which new vaccines can bedeveloped from lipid-containing cancer cells by partially removing thelipid envelope and exposing or modifying protein antigens hidden beneaththe envelope, in turn generating a positive immune response whenre-introduced, through various means, into the patient.

Cancers that may be treated with the present invention include all thosecancers known to physicians of ordinary skill in the medical arts,particularly physicians of skill in oncology. Cancers include, but arenot limited to, those arising from ectodermal, mesodermal and endodermalcells and include cancers of the immune system, the endocrine system,the central nervous system, the respiratory system, the reproductivesystem, the gastrointestinal system, and the integument. Such cancersinclude those generated by AIDS-related cancers, adrenocortical cancer,basal cell carcinoma, anal cancer, bladder cancer, bowel cancer, brainand central nervous system cancers, breast cancer, carcinoid cancers,cervical cancer, chondrosarcoma, choriocarcinoma, colorectal cancer,endocrine cancers, endometrial cancer, Ewing's sarcoma, eye cancer,gastric cancer, gastrointestinal cancer, genitourinary cancers, glioma,gynecological cancer, head and neck cancer, hepatocellular cancer,Hodgkin's disease, hypopharynx cancer, islet cell cancer, Kaposi'ssarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lungcancer, lymphoma, melanoma, mesothelioma, myeloma, nasopharyngealcancer, neuroblastoma, non-Hodgkin's lymphoma, esophagael cancer,osteosarcoma, ovarian cancer, pancreatic cancer, pituitary cancer, renalcell carcinoma, prostate cancer, retinoblastoma, rhabdomyosarcoma,sarcoma, skin cancer, squamous cell carcinoma, stomach cancer,testicular cancer, thymus cancer, thyroid cancer, transitional cellcancer, trophoblastic cancer, uterine cancer, vaginal cancer,Waldenstrom's macroglobulinemia, Wilm's cancer, among other tumors andforms of cancer.

Accordingly, it is an object of the present invention to provide avaccine composition comprising a cancer cell with reduced lipid contentand containing at least one cancer cell antigen in a pharmaceuticallyacceptable carrier and optionally an immunostimulant.

It is another object of the present invention to provide a vaccinecomposition comprising a cancer cell with reduced lipid content andcontaining at least one cancer cell antigen, and a dendritic cell, in apharmaceutically acceptable carrier, and optionally an immunostimulant.

Accordingly, it is an object of the present invention to provide amethod for treating a cancer cells in order to modify cancer cellscontained therein to reduce their lipid content.

It is a further object of the present invention to provide a method fortreating a cancer by administering cancer cells with reduced lipidcontent and containing at least one cancer cell antigen to an animal ora human.

Another object of the present invention is to provide a method forpreventing cancer or delaying the onset of cancer by administeringcancer cells with reduced lipid content and containing at least onecancer cell antigen to an animal or a human.

It is a further object of the present invention to provide a method fortreating a cancer using a DC-cancer cell hybrid exhibiting cancer cellantigens, wherein the cancer cell has a reduced lipid content andcontains at least one cancer cell antigen.

It is a further object of the present invention to provide a method forpreventing cancer or delaying the onset of cancer using a DC-cancer cellhybrid exhibiting cancer cell antigens.

It is another object of the present invention to provide a method forexposing antigenic determinants on a cancer cell.

It is a further object of the present invention to completely orpartially delipidate the cancer cell, thereby creating a modified cancercell with reduced lipid content and containing at least one cancer cellantigen.

It is a further object of the present invention to partially,substantially or completely delipidate the cancer cell, while retainingthe structural proteins or antigens of the cancer cell.

Yet another object of the present invention is to treat humans andanimals with cancer using the method of the present invention using avaccine comprising a DC-cancer cell hybrid wherein the cancer cell is apartially delipidated, modified cancer cell. The treatment may beadministered to an animal or a human together with a pharmaceuticallyacceptable carrier and optionally an immunostimulant compound.

Still another object of the present invention is to treat humans andanimals with cancer using the method of the present invention using avaccine comprising a cancer cell with reduced lipid content andcontaining at least one cancer cell antigen. The treatment may beadministered to an animal or a human together with a pharmaceuticallyacceptable carrier and optionally an immunostimulant compound.

Yet another object of the present invention is to treat humans andanimals at risk of developing cancer with the method of the presentinvention by administering a vaccine comprising a DC-cancer cell hybridwherein the cancer cell has reduced lipid content and contains at leastone cancer cell antigen. The treatment may be administered to an animalor a human together with a pharmaceutically acceptable carrier andoptionally an immunostimulant compound.

Still another object of the present invention is to treat humans andanimals at risk of developing cancer with the method of the presentinvention by administering a cancer cell with reduced lipid andcontaining at least one cancer cell antigen.

Yet another object of the present invention is to treat humans andanimals with cancer using the method of the present invention byadministering a vaccine comprising cancer cell-associated antigens froma cancer cell with reduced lipid content which may be administered to ananimal or a human together with a pharmaceutically acceptable carrierand optionally an immunostimulant compound, to initiate an immunogenicresponse in the animal or human.

Still another object of the present invention is to treat humans andanimals at risk of developing cancer using the method of the presentinvention by administering a vaccine comprising a cancer cell withreduced lipid content and containing at least one cancer cell antigenwhich may be administered to an animal or a human together with apharmaceutically acceptable carrier and optionally an immunostimulantcompound, to initiate an immunogenic response in the animal or human.

Yet another object of the present invention is to provide a method forpromoting antibody production comprising administering to the animal orhuman a cancer cell with reduced lipid content and containing at leastone cancer cell antigen together with a pharmaceutically acceptablecarrier in order to initiate an immunogenic response, resulting in theproduction of antibodies in the animal or human.

The present invention also provides a cancer cell with reduced lipidcontent and containing at least one cancer cell antigen, wherein thiscancer cell initiates an immune response when administered to a patientand incites protection against a cancer.

The present invention also provides for a patient-specific modifiedcancer cells comprising a partially delipidated cancer cell, wherein thepartially delipidated cancer cell is produced by exposing anon-delipidated cancer cell to a delipidation process and wherein thecancer cell with reduced lipid content comprises at least one exposed ormodified patient-specific antigen that was not exposed or modified inthe non-delipidated cancer cell.

The present invention also provides a method for making a vaccinecomprising: contacting a lipid-containing cancer cell in a fluid with afirst organic solvent capable of extracting lipid from thelipid-containing cancer cell; mixing the fluid and the first organicsolvent for a time sufficient to extract lipid from the lipid-containingcancer cell; permitting organic and aqueous phases to separate; andcollecting the aqueous phase containing a modified cancer cell withreduced lipid content wherein the modified cancer cell is capable ofprovoking a positive immune response when administered to a patient.

The present invention also provides a method for provoking a positiveimmune response in a patient having a plurality of lipid-containingcancer cells, comprising the steps of: obtaining a fluid containing thelipid-containing cancer cells from the patient; contacting the fluidcontaining the lipid-containing cancer cells with a first organicsolvent capable of extracting lipid from the lipid-containing cancercells; mixing the fluid and the first organic solvent: permittingorganic and aqueous phases to separate; collecting the aqueous phasecontaining modified cancer cell particles with reduced lipid content;and introducing the aqueous phase containing the modified cancer cellswith reduced lipid content into the animal or the human wherein themodified cancer cell with reduced lipid content provoke a positiveimmune response in the animal or the human.

Various modifications to the stated embodiments will be readily apparentto those of ordinary skill in the art, and the disclosure set forthherein may be applicable to other embodiments and applications withoutdeparting from the spirit and scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate preferred embodiments of the presentinvention.

FIG. 1 depicts uptake of delipidated B16-F10 cancer cells by immaturedendritic cells as determined by fluorescent activated cell sorting(FACS) (phycoerythrin (PE) labeled).

FIG. 2 depicts uptake of delipidated B16-F10 cancer cells by immaturedendritic cells as determined by FACS (fluorescein isothiocyanate)(FITC) labeled.

FIG. 3 depicts therapeutic vaccination and reduction of cancer growth.

FIG. 4 is similar to FIG. 3 and depicts therapeutic vaccination usingDCs pulsed with delipidated B16-F10 cancer cells.

FIG. 5 depicts preventative vaccination using delipidated B16-F10 cancercells.

FIG. 6 is similar to FIG. 5 and depicts preventative vaccination usingDCs and delipidated B16-F10 cancer cells.

FIG. 7 depicts therapeutic vaccination using delipidated B16-F10 cancercells to induce an antigen specific response.

FIG. 8 depicts therapeutic vaccination using delipidated B16-F10 cancercells to induce an antigen specific response.

FIG. 9 depicts therapeutic vaccination using DCs and delipidated B16-F10cancer cells to induce an antigen specific response.

FIG. 10 depicts therapeutic vaccination using DCs and delipidatedB16-F10 cancer cells to induce an antigen specific response.

FIG. 11 is a schematic of the therapeutic vaccination experimental plan.

FIG. 12 is a schematic of the preventative vaccination experimentalplan.

DETAILED DESCRIPTION OF THE INVENTION Definitions

By the term “fluid” is meant any fluid containing cancer cells,including but not limited to, a biological fluid obtained from anorganism such as an animal or human. Fluids which may be treated withthe method of the present invention include but are not limited to thefollowing: plasma; serum; lymphatic fluid; cerebrospinal fluid;peritoneal fluid; pleural fluid; pericardial fluid; various fluids ofthe reproductive system including but not limited to semen, ejaculatoryfluids, follicular fluid and amniotic fluid; cell culture reagents suchas normal sera, fetal calf serum or serum derived from any other animalor human; and immunological reagents such as various preparations ofantibodies and cytokines. Such biological fluids obtained from anorganism include but are not limited to other fluids contained withinthe organism. Other fluids may include laboratory samples containingcancer cells suspended in any chosen fluid. Other fluids include cellculture reagents, many of which include biological compounds such asfluids obtained from living organisms, including but not limited to“normal serum” obtained from various animals and used as growth mediumin cell and tissue culture applications.

By the term “first extraction solvent” is meant a solvent, comprisingone or more solvents, used to facilitate extraction of lipid from alipid-containing cell or a fluid. The term “first extraction solvent” isused interchangeably with “first organic solvent” in the presentapplication. This solvent will enter the fluid and remain in the fluiduntil being removed. Suitable first extraction solvents include solventsthat extract or dissolve lipid, including but not limited to alcohols,hydrocarbons, amines, ethers, and combinations thereof. First extractionsolvents may be combinations of alcohols and ethers. First extractionsolvents include, but are not limited to n-butanol, di-isopropyl ether(DIPE), diethyl ether, and combinations thereof. In another embodiment,the first extraction solvent may optionally include a detergent.

The term “second extraction solvent” is defined as one or more solventsthat may be employed to facilitate the removal of a portion of the firstextraction solvent. Suitable second extraction solvents include anysolvent that facilitates removal of the first extraction solvent fromthe fluid. Second extraction solvents include any solvent thatfacilitates removal of the first extraction solvent including but notlimited to ethers, alcohols, hydrocarbons, amines, and combinationsthereof. Preferred second extraction solvents include diethyl ether anddi-isopropyl ether, which facilitate the removal of alcohols, such asn-butanol, from the fluid. The term “de-emulsifying agent” is a secondextraction solvent that assists in the removal of the first extractionsolvent which may be present in an emulsion in an aqueous layer. By theterm “de-emulsifying agent” is meant an agent that assists in theremoval of the first extraction solvent which may be present in anemulsion in an aqueous layer.

Detergents and surfactants known to one of ordinary skill in the art maybe employed in combination with the at least first extraction solvent inthe present invention. Such detergents and surfactants include, but arenot limited to, various ionic and non-ionic detergents. Such detergentsand surfactants include but are not limited to various forms of Tritonor Tween.

The terms “modified cancer cell” and “cancer cell particle” are usedinterchangeably and describe a modified cancer cell, cancer cellparticle or fragments thereof that results from application of theprocess of the present invention to cancer cells in order to reducetheir lipid content.

The term “delipidation” refers to the process of removing at least aportion of a total concentration of lipids from a cancer cell.

The term “lipid” is defined as any one or more of a group of fats orfat-like substances occurring in humans or animals. The fats or fat-likesubstances are characterized by their insolubility in water andsolubility in organic solvents. The term “lipid” is known to those ofordinary skill in the art and includes, but is not limited to, polarlipids, non-polar lipids, complex lipid, simple lipid, triglycerides,fatty acids, glycerophospholipids (phospholipids), sphingolipids, truefats such as esters of fatty acids, glycerol, cerebrosides, waxes, andsterols such as cholesterol and ergosterol. Lipids which can be removedfrom a cancer cell include but are not limited to the removal of polarlipids, non-polar lipids, sphingolipids, cholesterol, phospholipids or acombination thereof. In one embodiment, the total concentration of lipidremaining in the modified cancer cell is less than 80% of the totalconcentration of lipid in the original cancer cell. In anotherembodiment, the total concentration of lipid remaining in the modifiedcancer cell is less than 50% of the total concentration of lipid in theoriginal cancer cell. In a preferred embodiment, the total concentrationof lipid remaining in the modified cancer cell is less than 30% of thetotal concentration of lipid in the original cancer cell. In a furtherembodiment, the total concentration of lipid remaining in the modifiedcancer cell is less than 20% of the total concentration of lipid in theoriginal cancer cell. In a further embodiment, the total concentrationof lipid remaining in the modified cancer cell is less than 10% of thetotal concentration of lipid in the original cancer cell. In a furtherembodiment, the total concentration of lipid remaining in the modifiedcancer cell is less than 5% of the total concentration of lipid in theoriginal cancer cell. In another embodiment, the total concentration oflipid remaining in the modified cancer cell is between 1% and 80% of thetotal concentration of lipid in the original cancer cell.

Additionally, modified cancer cells and cancer cell particles may in oneembodiment possess protein recovery rates in excess of 50% of the totalprotein content as compared to a non-delipidated cancer cell.

The terms “pharmaceutically acceptable carrier or pharmaceuticallyacceptable vehicle” are used herein to mean any liquid including but notlimited to water or saline, a gel, salve, solvent, diluent, fluidointment base, liposome, micelle, giant micelle, and the like, which issuitable for use in contact with living animal or human tissue withoutcausing adverse physiological responses, and which does not interactwith the other components of the composition in a deleterious manner.

The term “patient” refers to an animal or a human.

The term “patient specific antigen” refers to an antigen that is capableof inducing a patient specific immune response when introduced into thatpatient. Such patient specific antigens may be cancer associatedantigens or tumor associated antigens. A patient specific antigenincludes any antigen, for example a cancer associated antigen.

The term “Tumor Associated Antigen (TAA) or cancer cell associatedantigen refers to an antigen known to one of ordinary skill in the artas being associated with a tumor or cancer cell. Non-limiting examplesof TAAs embodied by the present invention can be found in Table 1. Itwill be apparent to one of ordinary skill in the art that a TAA cancomprise a cell surface antigen, membrane bound to a cancer cell. Manysuch antigens are glycosylated, for example GP-100. In anotherembodiment, the TAA's can also comprise intracellular cancer specificantigens. As demonstrated herein, DCs can uptake and process delipidatedcancer cells. Accordingly, the present invention encompasses a methodfor the presentation of intracellular cancer specific antigens to thecell-mediated immune system.

TABLE 1 Tumor/Cancer Associated Antigens (TAAs) embodied by the instantapplication. AFP Alpha (α)-fetoprotein AIM-2 Interferon-inducibleprotein absent in melanoma 2 ALL Acute lymphoblastic leukemia AML Acutemyeloid leukemia 707-AP 707 alanine proline APL Acute promyelocyticleukemia ART-4 Adenocarcinoma antigen recognized by T cells 4 BAGE Bantigen bcr-abl Breakpoint cluster region- Abelson CAMEL CTL-recognizedantigen on melanoma CAP-1 Carcinoembryonic antigen peptide-1 CASP-8Caspase 8 CDC27 Cell division cycle 27 CDK4 Cyclin-dependent kinase 4CEA Carcinoembryonic antigen CLCA2 Calcium-activated chloride channel 2CML Chronic myelogenous leukemia CT Cancer-testis (antigen) CTLCytotoxic T lymphocytes Cyp-B Cyclophilin B DAM Differentiation antigenmelanoma ELF2 Elongation factor 2 Ep-CAM Epithelial cell adhesionmolecule EphA2, 3 Ephrin type-A receptor 2, 3 Ets E-26 transformingspecific ETV6-AML1 Ets variant gene 6/acute myeloid leukemia 1 gene ETSFGF-5 Fibroblast growth factor 5 FN Fibronectin G250 Glycoprotein 250GAGE G antigen GnT-V N-Acetylglucosaminyltransferase V Gp100Glycoprotein 100 kDa HAGE Helicase antigen HER-2/neu Human epidermalreceptor 2/ neurological HAL-A*0201- Arginine (R) to isoleucine (I)R170I exchange at residue 170 of the α-helix of the α2-domain in theHLA-A2 gene H/N Head and neck HSP70-2M Heat shock protein 70-2 mutatedHST-2 Human signet-ring tumor 2 hTERT Human telomerase reversetranscriptase iCE Intestinal carboxyl esterase IL-13Rα2 Interleukin 13receptor α2 chain KIAA0205 Name of the gene as it appears in databasesLAGE L antigen LDLR/FUT Low density lipid receptor/GDP-L-fucose:β-D-galactosidase 2-α-L-fucosyltransferase MAGE Melanomaantigen MART-1/Melan-A Melanoma antigen recognized by T cells-1/melanomaantigen A MART-2 Melanoma Ag recognized by T cells-2 MCIR Melanocortin 1receptor M-CSF Macrophage colony-stimulating factor gene MHC Majorhistocompatibility complex MSI Microsatellite instability MUC1, 2 Mucin1, 2 MUM-1, -2, -3 Melanoma ubiquitous mutated 1, 2, 3 NA88-A NA cDNAclone of patient M88 Neo-PAP Neo-poly(A) polymerase NPM/ALKNucleophosmin/anaplastic lymphoma kinase fusion protein NSCLC Non-smallcell lung carcinoma NY-ESO-1 New York esophageous 1 OA1 Ocular albinismtype 1 protein OGT O-Linked N-acetylglucosamine transferase gene ORFOpen reading frame OS-9 Name of the gene as it appear in databases P15Protein 15 p190 mnor bcr-abl Protein of 190-kDa bcr-abl Pml/RARαPromyelocytic leukemia/ retinoic acid receptor α PRAME Preferentiallyexpressed antigen of melanoma PSA Prostrate-specific antigen PSMAProstrate-specific membrane antigen PTPRK Receptor-type protein-tyrosinephosphatase kappa RAGE Renal antigen RCC Renal cell carcinoma RU1, 2Renal ubiquitous 1, 2 SAGE Sarcoma antigen SART-1, -2, -3 Squamousantigen rejecting tumor 1, 2, 3 SCC Squamous cell carcinoma SSX-2Synovial sarcoma, X breakpoint 2 Survivin-2B Intron 2-retaining survivinSYT/SSX Synaptotagmin I/synovial sarcoma, X fusion protein TEL/AML1Translocation Ets-family leukemia/acute myeloid leukemia 1 TGFβRIITransforming growth factor β receptor 2 TPI Triosephosphate isomeraseTRAG-3 TAxol resistant associated protein 3 TRG Testin-related geneTRP-1 Tyrosinase-related protein 1, or gp75 TRP-2 Tyrosinase-relatedprotein 2 TRP-2/INT2 TRP-2/intron 2 TRP-2/6B TRP-2/novel exon 6b TSTATumor-specific transplantation antigens WT1 Wilms' tumor gene

Methods of Manufacture of the Modified Cancer Cell

One of ordinary skill in the art would appreciate that multipledelipidation processes are encompassed within the scope of the presentinvention. In a preferred embodiment, a solvent system together with amechanical mixing system is used to substantially delipidate the cancercell. The delipidation process is dependent upon the total amount ofsolvent and energy input into a system. Various solvent levels andmixing methods, as described below, may be used depending upon theoverall framework of the process. Practice of the method of the presentinvention to reduce the lipid content of a cancer cell creates amodified cancer cell or cancer cell particle. These modified cancer cellhave lower levels of lipid and are immunogenic. The present methodsexpose or modify epitopes that are not usually presented to the immunesystem by untreated cancer cells. It is believed that delipidation notonly exposes epitopes but also enhances the antigen processing andpresentation of tumor associated antigens because of the conformationalshape the antigen is presented in after delipidation. Methods of thepresent invention solve numerous problems encountered with prior artmethods. By substantially decreasing the lipid content of the lipidenvelope of the cancer cell, and keeping the modified cancer cellintact, the method of the present invention exposes or modifiesadditional antigens. The host immune system recognizes the modifiedcancer cell as foreign. Using the method of the present invention, whatis created is a modified cancer cell or cancer cell particle in whichadditional antigens are exposed, thereby using the epitopes of theactual cancer cell to initiate a positive immunogenic response in thepatient following administration.

Modified, partially delipidated cancer cells or particles obtained withsome embodiments of the methods disclosed herein represent, in someaspects, new therapeutic vaccine compositions for therapeuticimmunization and induction of an immune response in animals or humans.In one aspect, modified, partially delipidated cancer cell obtained withthe methods disclosed herein are useful for therapeutic immunization andinduction of an immune response in animals or humans afflicted with acancer. In another embodiment, modified cancer cells obtained with themethods disclosed herein are useful for immunization of animals andhumans who do not have the cancer in order to provoke an immune responsewhen cancers may develop. In one embodiment of the present invention,administration of the modified, partially delipidated cancer cells andcompositions comprising such cells provides a new method of treatment,alleviation, or containment of cancer growth, conditions or clinicalsymptoms associated with the cancer.

Partially delipidated cancer cells and cancer cell particles obtainedaccording to some of aspects of the present invention possess at leastsome structural characteristics that distinguish them from conventionalcancer cells. Such characteristics include, but are not limited to,reduced lipid content, modified protein content, or the ratio of lipidcontent to protein content. For example, a partially delipidated cancercell or cancer cell particle according to some embodiments of thepresent invention has a lower cholesterol content than the cholesterolcontent of the non-delipidated cancer cell. In one embodiment, the lowercholesterol content of the partially delipidated cancer cell particlecan be at least 70% to 99% lower than the cholesterol content ofnon-delipidated cancer cells. In other embodiments, the cholesterolcontent in the modified, partially delipidated cancer cell particle isreduced, for example, by about 99%, 90%, 70%, 50%, 30% or 20% ascompared to the unmodified cancer cell. It is to be understood thatcholesterol is but one form of lipid which may be reduced followingtreatment of the cancer cells, and other lipids as defined herein, orcombinations of these lipids may be reduced.

Modified, partially delipidated cancer cell may also be characterized,for example, as retaining >50% of the cancer cell total protein content.In one embodiment of the present invention, the TAAs that are retainedin the modified cancer cell include but are not limited to TAAs found inTable 1. In another embodiment the TAAs comprise GP 100 and TRP-2.However, it will be appreciated by one of ordinary skill in the art thatthe TAAs retained by the modified cancer cell will vary depending on thetype of cancer present.

Exemplary Solvent Systems for use in Removal of Lipid from Cancer Cellsto Produce Delipidated Cancer Cells or Particles Useful for VaccineProduction

The solvent or combinations of solvents to be employed in the process ofpartially or completely delipidating lipid-containing cancer cells andin producing vaccines may be any solvent or combinations thereofeffective in solubilizing lipids while retaining antigen components ofthe cancer cell, which can be measured in one embodiment, via proteinrecovery. This delipidation process that keeps antigen components of thecancer cell intact is a matter of defining the right solvent-energysystems. Suitable solvents comprise hydrocarbons, ethers, alcohols,phenols, esters, halohydrocarbons, halocarbons, amines, and mixturesthereof. Aromatic, aliphatic, or alicyclic hydrocarbons may also beused. Other suitable solvents, which may be used with the presentinvention, include amines and mixtures of amines. A preferred solventcombination comprises alcohols and ethers. One solvent system is DIPE,either concentrated or diluted in water or a buffer such as aphysiologically acceptable buffer. One solvent combination comprisesalcohols and ethers. Another preferred solvent comprises ether orcombinations of ethers, either in the form of asymmetrical ethers orhalogenated ethers.

The optimal solvent systems are those that accomplish two objectives:first, at least partially delipidating the cancer cell and second,providing few or no deleterious effects on the antigenic proteins of thecancer cells. In addition, the solvent system should maintain theintegrity of the cancer cell particle such that it can be used toinitiate an immune response in the patient. It should therefore be notedthat certain solvents, solvent combinations, and solvent concentrationsmay be too harsh to use in the present invention because they result inunacceptable degradation of cancer cell proteins.

It is preferred that the solvent or combination of solvents has arelatively low boiling point to facilitate removal through a vacuum andpossibly heat without destroying the antigens of the cancer cell. It isalso preferred that the solvent or combination of solvents be employedat a low temperature because heat may have deleterious effects onproteins. It is also preferred that the solvent or combination ofsolvents at least partially delipidate the cancer cell.

Removal of solvents from delipidated cancer cells may be accomplishedthrough use of a second extraction solvent or a de-emulsifying agent.For example, demulsifying agents such as ethers may be used to remove afirst solvent such as an alcohol from an emulsion. Removal of solventsmay also be accomplished through other methods, which do not employadditional solvents, including but not limited to the use of charcoal.Charcoal may be used in a slurry or alternatively, in a column to whicha mixture is applied. Charcoal is a preferred method of removingsolvents. Pervaporation may also be employed to remove one or moresolvents from delipidated cancer cell mixtures.

Examples of suitable amines for use in removal of lipid fromlipid-containing cancer cells in the present invention are those whichare substantially immiscible in water. Typical amines are aliphaticamines—those having a carbon chain of at least 6 carbon atoms. Anon-limiting example of such an amine is C₆H₁₃NH₂.

The preferred alcohols for use in the present invention, when usedalone, include those alcohols that are not appreciably miscible withplasma or other biological fluids. Such alcohols include, but are notlimited to, straight chain and branched chain alcohols, includingpentanols, hexanols, heptanols, octanols and those alcohols containinghigher numbers of carbons. Alcohols may be used alone or in combinationwith another solvent, for example an ether. Concentrations of alcoholsmay be employed to remove lipids when used alone and not in combinationwith other solvents. For example, a concentration range of alcoholsinclude 0.1% to 99.9%. For example, concentrations of alcohols that maybe employed include, but are not limited to the following: 0.1%, 1.0%,2.5%, 5%, 10.0% and 25% or higher.

When alcohols are used in combination with another solvent, for example,an ether, a hydrocarbon, an amine, or a combination thereof, C₁-C₈containing alcohols may be used. Preferred alcohols for use incombination with another solvent include C₄-C₈ containing alcohols.Accordingly, preferred alcohols that fall within the scope of thepresent invention are butanols, pentanols, hexanols, heptanols andoctanols, and iso forms thereof. In particular, C₄ alcohols or butanols(1-butanol and 2-butanol) are preferred. The specific alcohol choice isdependent on the second solvent employed. In a preferred embodiment,lower alcohols are combined with lower ethers.

Ether, when used either alone or in combination with other solvents(preferably alcohols), is another preferred solvent for use in themethod of the present invention. Particularly preferred are the C₄-C₈containing-ethers, including but not limited to ethyl ether, diethylether, and propyl ethers (including but not limited to di-isopropylether (DIPE)). Asymmetrical ethers may also be employed. Halogenatedsymmetrical and asymmetrical ethers may also be employed.

Low concentrations of solvents, such as ethers, may be employed toremove lipids when used alone and not in combination with othersolvents. For example, a low concentration range of ethers include 0.5%to 30%. For example, concentrations of ethers that may be employedinclude, but are not limited to the following: 0.625%, 1.0% 1.25%, 2.5%,3%, 5.0% and 10% or higher. It has been observed that dilute solutionsof ethers are effective to remove lipids from cells. Such solutions maybe aqueous solutions or solutions in aqueous buffers, such as phosphatebuffered saline (PBS). Other physiological buffers may be used,including but not limited to bicarbonate, citrate, Tris, Tris/EDTA, andTrizma. Preferred ethers are di-isopropyl ether (DIPE) and diethyl ether(DEE). Ethers may also be used in combination in the presentinvention—such as a solvent mixture of DIPE and DEE. Low concentrationsof ethers may also be used in combination with alcohols, for example,n-butanol.

When ethers and alcohols are used in combination as a first solvent forremoving lipid from lipid-containing cancer cells, any combination ofalcohol and ether may be used provided the combination is effective toat least partially remove lipid from the cancer cell, without havingdeleterious effect on the immunogenic proteins. When alcohols and etherare combined as a first solvent for treating the cancer cells containedin a fluid, useful ratios of alcohol to ether in this solvent range fromabout 0.01 parts alcohol to 99.99 parts ether to 60 parts alcohol to 40parts ether, with a specific ratio range of about 10 parts alcohol to 90parts ether to 5 parts alcohol to 95 parts ether, with a specific ratiorange of about 10 parts alcohol to 90 parts ether to 50 parts alcohol to50 parts ether, with a specific ratio range of about 20 parts alcohol to80 parts ether to 45 parts alcohol to 55 parts ether, with a specificrange of about 25 parts alcohol to 75 parts ether with respect to eachother. In one embodiment, the ratio of alcohol to ether is 1 partalcohol, to 1 part ether and 98 parts fluid containing the cancer cells.

An especially preferred combination of alcohol and ether is thecombination of butanol and DIPE. When butanol and DIPE are combined as afirst solvent for treating cancers contained in a fluid, useful ratiosof butanol to DIPE in this solvent are about 0.01 parts butanol to 99.99parts DIPE to 60 parts butanol to 40 parts DIPE, with a specific ratiorange of about 10 parts butanol to 90 parts DIPE to 5 parts butanol to95 parts DIPE, with a specific ratio range of about 10 parts butanol to90 parts DIPE to 50 parts butanol to 50 parts DIPE, with a specificratio range of about 20 parts butanol to 80 parts DIPE to 45 partsbutanol to 55 parts DIPE, with a specific range of about 25 partsbutanol to 75 parts DIPE with respect to each other. In anotherembodiment, a ratio range of combined solvent to tumor cell containingfluid are about 0.5 parts combined solvent to 99.5 parts tumor cellcontaining fluid to 2 parts combined solvent to 1 part cancer cellcontaining fluid.

Another combination of alcohol and ether is the combination of butanolwith DEE. When butanol is used in combination with DEE as a firstsolvent, useful ratios of butanol to DEE are about 0.01 parts butanol to99.99 parts DEE to 60 parts butanol to 40 parts DEE, with a specificratio range of about 10 parts butanol to 90 parts DEE to 5 parts butanolto 95 parts DEE with a specific ratio range of about 10 parts butanol to90 parts DEE to 50 parts butanol to 50 parts DEE, with a specific ratiorange of about 20 parts butanol to 80 parts DEE to 45 parts butanol to55 parts DEE, with a specific range of about 40 parts butanol to 60parts DEE.

Additionally, when employing a solvent containing n-butanol, the presentinvention can also use a ratio of solvent that yields about 0.1%-5%n-butanol in the final solvent/cancer cell suspension, for example,0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% n-butanol may be used.

Liquid hydrocarbons dissolve compounds of low polarity such as thelipids found in the cancer cells. Particularly effective in disruptingthe lipid membrane of a cancer cell are hydrocarbons which aresubstantially water immiscible and liquid at about 37° C. Suitablehydrocarbons include, but are not limited to the following: C₅ to C₂₀aliphatic hydrocarbons such as petroleum ether, hexane, heptane, octane;haloaliphatic hydrocarbons such as chloroform,1,1,2-trichloro-1,2,2-trifluoroethane, 1,1,1-trichloroethane,trichloroethylene, tetrachloroethylene, dichloromethane and carbontetrachloride; thioaliphatic hydrocarbons each of which may be linear,branched or cyclic, saturated or unsaturated; aromatic hydrocarbons suchas benzene; alkylarenes such as toluene; haloarenes; haloalkylarenes;and thioarenes. Other suitable solvents may also include saturated orunsaturated heterocyclic compounds such as pyridine and aliphatic, thio-or halo-derivatives thereof.

Suitable esters for use in the present invention include, but are notlimited to, ethyl acetate, propylacetate, butylacetate andethylpropionate. Suitable detergents/surfactants that may be usedinclude but are not limited to the following: sulfates, sulfonates,phosphates (including phospholipids), carboxylates, and sulfosuccinates.Some anionic amphiphilic materials useful with the present inventioninclude but are not limited to the following: sodium dodecyl sulfate(SDS), sodium decyl sulfate, bis-(2-ethylhexyl) sodium sulfosuccinate(AOT), cholesterol sulfate and sodium laurate.

Cancer Cells and Treatment thereof for Producing Exposed CancerCell-Associated Antigens

As stated above, various cancers may be treated with the method of thepresent invention in order to expose cancer cell-associated antigens ofthe cancer cells. In a preferred embodiment, cancer samples obtainedfrom an animal or human are treated with the method of the presentinvention in order to remove lipid and expose or modify cancercell-associated antigens. In this embodiment, cancer samples such asbiopsies may be obtained from an animal or human patient by anyconventional means, including various surgical techniques and treatingthe sample in order to isolate the cancer cells. Some cancer cells maybe obtained from fluids such as plasma, peritoneal, pleural, pericardialand cerebrospinal fluids. Such methods for excising and isolating cancercells are known to one of ordinary skill in the art.

Once a cancer cell is obtained either in this manner, or for example,from a storage facility housing samples of cancer cells, the cancer cellis contacted with a first organic solvent, as described above, capableof solubilizing lipid in the cancer cell. The first organic solvent iscombined with the cancer cells or a medium containing the cancer cellsin a ratio wherein the first solvent is present in an amount effectiveto substantially solubilize the lipid in the cancer cells, for example,dissolve the lipid envelope that surrounds the cells. Acceptable ratiosof first solvent to medium (expressed as a ratio of first organicsolvent to the medium containing the cancer cells) are described in thefollowing ranges: 0.5-4.0:0.5-4.0; 0.8-3.0:0.8-3.0; and 1-2:0.8-1.5.Various other ratios may be applied, depending on the nature of themedium and concentration of the cancer cells in that medium. Forexample, in the case of cell culture fluid, the following ranges may beemployed of first organic solvent to cell culture fluid:0.5-4.0:0.5-4.0; 0.8-3.0:0.8-3.0; and 1-2:0.8-1.5.

After contacting the medium containing the cancer cells with the firstsolvent as described above, the first solvent and medium are mixed usinga method that includes, but is not limited to, any one of the followingsuitable mixing methods: gentle stirring; vigorous stirring; vortexing;swirling; shaking, homogenization; and end-over-end rotation. In oneembodiment, the first solvent and medium are mixed using end-over-endrotation. In another embodiment, the first solvent and medium are mixedby shaking.

The amount of time required for adequate mixing of the first solventwith the medium is related to the mixing method employed. Medium ismixed for a period of time sufficient to permit intimate contact betweenthe organic and aqueous phases, and for the first solvent to at leastpartially or completely solubilize the lipid contained in the cancercells. Typically, mixing will occur for periods of about 5 seconds toabout 24 hours, 10 seconds to about 2 hours, approximately 10 seconds toapproximately 10 minutes, or about 30 seconds to about 1 hour, dependingon the mixing method employed and the quantity of the cells beingtreated. Non-limiting examples of mixing durations associated withdifferent methods are presented in the next sentences. Gentle stirringand end-over-end rotation may occur for a period of about 5 seconds toabout 24 hours. Vigorous stirring and vortexing may occur for a periodof about 5 seconds to about 30 minutes. Swirling may occur for a periodof about 5 seconds to about 2 hours. Homogenization may occur for aperiod of about 5 seconds to about 10 minutes. Shaking may occur for aperiod of about 5 seconds to about 2 hours.

Separation of Solvents

After mixing the first solvent with the medium, the solvent is separatedfrom the medium being treated. The organic and aqueous phases may beseparated by any suitable manner known to one of ordinary skill in theart. Since the first solvent is typically immiscible in the aqueousfluid, the two layers are permitted to separate and the undesired layeris removed. The undesired layer is the solvent layer containingdissolved lipids and its identification, as known to one of ordinaryskill in the art, depends on whether the solvent is more or less densethan the aqueous phase. An advantage of separation in this manner isthat dissolved lipids in the solvent layer may be removed.

In addition, separation may be achieved through means, including but notlimited to the following: removing the undesired layer via pipetting;centrifugation followed by removal of the layer to be separated;creating a path or hole in the bottom of the tube containing the layersand permitting the lower layer to pass through; utilization of acontainer with valves or ports located at specific lengths along thelong axis of the container to facilitate access to and removal ofspecific layers; and any other means known to one of ordinary skill inthe art. Another method of separating the layers, especially when thesolvent layer is volatile, is through distillation under reducedpressure or evaporation at room temperature, optionally combined withmild heating. In one embodiment employing centrifugation, relatively lowg forces are employed, such as 900× g for about 5 to 15 minutes toseparate the phases.

A preferred method of removing solvent is through the use of charcoal,preferably activated charcoal. This charcoal is optionally contained ina column. Alternatively the charcoal may be used in slurry form. Variousbiocompatible forms of charcoal may be used in these columns.Pervaporation methods and use of charcoal to remove solvents arepreferred methods for removing solvent.

Following separation of the first solvent from the treated medium, someof the first solvent may remain entrapped in the aqueous layer as anemulsion. Optionally, a de-emulsifying agent is employed to facilitateremoval of the trapped first solvent. Still another method of removingsolvent is the use of hollow fiber contactors. The de-emulsifying agentmay be any agent effective to facilitate removal of the first solvent. Apreferred de-emulsifying agent is ether and a more preferredde-emulsifying agent is diethyl ether. The de-emulsifying agent may beadded to the fluid or in the alternative the fluid may be dispersed inthe de-emulsifying agent. In vaccine preparation, alkanes in a ratio ofabout 0.5 to 4.0 to about 1 part of emulsion (vol:vol) may be employedas a demulsifying agent, followed by washing to remove the residualalkane from the remaining delipidated cancer cell used for preparing thevaccine. Preferred alkanes include, but are not limited to, pentane,hexane and higher order straight and branched chain alkanes.

The de-emulsifying agent, such as ether, may be removed through meansknown to one of skill in the art, including such means as described inthe previous paragraph. One convenient method to remove thede-emulsifying agent, such as ether, from the system, is to permit theether to evaporate from the system in a running fume hood or othersuitable device for collecting and removing the de-emulsifying agentfrom the environment. In addition, de-emulsifying agents may be removedthrough application of higher temperatures, for example from about 24 to37° C. with or without pressures of about 10 to 20 mbar. Another methodto remove the de-emulsifying agent involves separation bycentrifugation, followed by removal of organic solvent throughaspiration, further followed by evaporation under reduced pressure (forexample 50 mbar) or further supply of an inert gas, such as nitrogen,over the meniscus to aid in evaporation.

Cancer cells or fragments of cancer cells treated with the delipidationmethod of the present invention may be collected or concentrated usingmethods known to one of ordinary skill in the art. Such methods includebut are not limited to the following, centrifugation, filtration,sieving, cell sorting and chromatography, for example affinitychromatography.

Methods of Treating Biological Fluids Containing Cancer Cells(Delipidation)

It is to be understood that the method of the present invention isemployed in either a continuous or discontinuous manner. In adiscontinuous or batch mode of operation, the present invention employsa cancer tissue sample or dispersed cells previously obtained from ahuman or animal. The sample is treated with the method of the presentinvention to produce a new sample which contains at least partially orcompletely delipidated cancer cells, or modified cancer cells. Oneembodiment of this mode of the present invention is to treat cancer cellsamples previously obtained from animals or humans and stored in a cellbank for subsequent use to create DC-cancer cell hybrids. These samplesmay be administered with the method of the present invention toeliminate cancers or minimize the proliferation of a cancer.

Delipidation of cancer cells can be achieved by various means. A batchmethod can be used for fresh or stored cancer cells. In this case avariety of the described organic solvents or mixtures thereof can beused for cancer cell delipidation. Extraction time depends on thesolvent or mixture thereof and the mixing procedure employed.

Through the use of the methods of the present invention, levels of lipidin lipid-containing cancer cells are reduced, and the fluid, forexample, containing the delipidated cancer cell particles may beadministered to the patient. Such fluid containing modified cancer cellparticles may act as a vaccine and provide protection in the patientagainst the cancer or provide a treatment in a patient afflicted withthe cancer by generating an immune response and decreasing the severityof the cancer. These modified cancer particles induce an immune responsein the recipient to exposed epitopes on the modified cancer cellparticles. Alternatively the modified cancer cell particles may becombined with a pharmaceutically acceptable carrier, and optionally anadjuvant, and administered as a vaccine composition to a human or ananimal to induce an immune response in the recipient.

Vaccine Production—Embodiment One

The modified cancer cell, which is at least partially or substantiallydelipidated and has exposed tumor associated antigens, has immunogenicproperties and is combined with a pharmaceutically acceptable carrier tomake a composition comprising a vaccine. This vaccine composition isoptionally combined with an adjuvant or an immunostimulant andadministered to an animal or a human. Both autologous and non-autologousvaccines, including combination vaccines, are within the scope of thepresent invention. It is to be understood that vaccine compositions maycontain more than one type of modified cancer cell or component thereof,in order to provide protection against complex cancers. Suchcombinations may be selected according to the desired immunity.

Vaccine Production Employing Dendritic Cells and Delipidated CancerCells—Embodiment Two

Dendritic cells can be used to induce an antitumoral response within apatient. Dendritic cells are hematopoietically derived leucocytes thatform a cellular network involved in immune surveillance, antigencapture, and antigen presentation. There are numerous techniques forisolating and propagating DCs in vitro known to one of ordinary skill inthe art (see: M. B. Lutz et al. J. Imm. Methods 223(1999) 9277-9279)and, therefore, they can be used in immunization strategies. To date,DCs together with synthetic peptides having known cancer antigens,stripped peptides derived from class I molecules, tumor RNA, or tumorlysates have been used to improve the immunogenic response of patientsto cancer.

In one embodiment, tumor tissue is removed, delipidated in accordancewith the above-described invention, placed in phosphate-buffered saline(PBS), and used to produce a single-cell suspension. Cells are lysedusing techniques known to one of ordinary skill in the art, for exampleby multiple freeze cycles, such as three to five, in liquid nitrogen andthaw cycles at room temperature. Lysis is preferably monitored. Largeparticles are removed by centrifugation and supernatants are passedthrough a filter. The protein contents are determined and stored forfuture use. The objective of this lysis step is to produce antigeniccomponents that are exposed via the delipidation process.

After generating dendritic cells from peripheral blood, in accordancewith methods known to those of ordinary skill in the art, the dendriticcells are cultured, i.e., for about 7 days, and then further culturedwith keyhole limpet hemocyanin (KLH) and the cancer cell lysate. One ofordinary skill in the art would recognize that the relative amounts ofKLH, tumor lysate, and DCs is dependent upon, and relative to, the kindof tumor to be treated. The resultant cells are washed with PBS and thenresuspended in RPMI-1640 for use in treatment. In one embodiment, thecell vaccine preparation is prepared in a solution suitable foradministration to humans, such as, but not limited to, saline, PBS orother approved solutions. This process creates a DC-cancer cell hybrid.

It is to be understood that other molecules besides KLH may be used, forexample, thyroglobulin or serum albumin, as commonly known to one ofordinary skill in the art.

Administration of Vaccine of Embodiment One, Produced with the Method ofthe Present Invention

When a delipidated cancer cell is administered to an animal or a human,it is typically combined with a pharmaceutically acceptable carrier toproduce a vaccine, and optionally combined with an adjuvant or animmunostimulant as known to one of ordinary skill in the art. Thevaccine formulations may conveniently be presented in unit dosage formand may be prepared by conventional pharmaceutical techniques known toone of ordinary skill in the art. Such techniques include uniformly andintimately bringing into association the active ingredient and theliquid carriers (pharmaceutical carrier(s) or excipient(s)).Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations may be presented in unit-dose or multi-dosecontainers—for example, sealed ampules and vials—and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water for injections, immediatelyprior to use. The vaccine may be stored at temperatures of from about 4°C. to −100° C. The vaccine may also be stored in a lyophilized state atdifferent temperatures including room temperature. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets commonly used by one of ordinary skill inthe art. The vaccine may be sterilized through conventional means knownto one of ordinary skill in the art. Such means include, but are notlimited to filtration, radiation and heat. The vaccine of the presentinvention may also be combined with bacteriostatic agents, such asthimerosal, to inhibit bacterial growth.

Preferred unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients, particularlymentioned above, the formulations of the present invention may includeother agents commonly used by one of ordinary skill in the art.

The vaccine may be administered through different routes, such as oral,including buccal and sublingual, rectal, parenteral, aerosol, nasal,intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal,and topical. The vaccine may also be administered in the vicinity oflymphatic tissue, for example through administration to the lymph nodessuch as axillary, inguinal or cervical lymph nodes, or through thelymphatic tissue of the gut (GALT).

The vaccine of the present invention may be administered in differentforms, including but not limited to solutions, emulsions andsuspensions, microspheres, particles, microparticles, nanoparticles, andliposomes. It is expected that from about 1 to 5 dosages may be requiredper immunization regimen. Initial injections may range from about 1 ngto 1 gram, from about 0.1 mg to 800 mg, and from approximately 1 mg to500 mg. Booster injections may range from 1 ng to 1 gram, fromapproximately 0.1 mg to 750 mg, and from about 0.5 mg to 500 mg. Thevolume of administration will vary depending on the administrationroute. Intramuscular injections may range from about 0.01 ml to 1.0 ml.

One of ordinary skill in the medical or veterinary arts of administeringvaccines will be familiar with the amount of vaccine to be administeredin an initial injection and in booster injections, if required, takinginto consideration, for example, the age and size of a patient. Initialinjections may range from about less than 1 ng to 1 gram based on totalcancer cell protein. A non-limiting range may be 1 ml to 10 ml. Thevolume of administration may vary depending on the administration route.

The vaccines of the present invention may be administered afterdetecting cancer. The vaccine of the present invention may beadministered to either humans or animals. In one embodiment, theproliferation of a cancer may be reduced by introducing delipidatedcancer cells. In another embodiment, the size of the cancer is actuallyreduced by introducing delipidated cancer cells.

In another embodiment, the vaccines of the present invention may beadministered to an individual without cancer or at high risk ofdeveloping cancer in order to prevent or delay the onset of cancer. Forexample, women with mothers and/or grandmothers who had ovarian cancerand/or breast cancer are more likely to develop ovarian cancer or breastcancer. Other sex-linked cancers are known to one of ordinary skill inthe art, and oncologists routinely advise patients that they may be morelikely to develop cancer based on family history, or the interaction offamily history and predisposing environmental or life style factors.Administration of the vaccines of the present invention, for example avaccine against ovarian cancer, to an individual at risk of developingovarian cancer, delays or prevents the occurrence of ovarian cancer inthat individual.

Administration of Vaccine of Embodiment Two, Produced with the Method ofthe Present Invention

After generating the DC-cancer lysates, as described above, it istypically combined with a pharmaceutically acceptable carrier to producea vaccine, and optionally combined with an adjuvant or animmunostimulant as known to one of ordinary skill in the art. Thevaccine formulations may conveniently be presented in unit dosage formand may be prepared by conventional pharmaceutical techniques known toone of ordinary skill in the art. Such techniques include uniformly andintimately bringing into association the active ingredient and theliquid carriers (pharmaceutical carrier(s) or excipient(s)).Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

One preferred approach to administering DC therapy includes intradermaladministration or administration directly into lymph nodes. In oneexemplary embodiment, patients receive the DCs pulsed with autologouscancer lysate every 3 weeks for a minimum of one and a maximum of 10immunizations. For example, patients receive four vaccinations at 3 weekintervals. Immunizations continue depending upon clinical response. Inone embodiment, dendritic cells injected per vaccination range from10×10⁵ to 32×10⁶ cells. However, it will be appreciated by one ofordinary skill in the art that the number of cells is variable dependingon the type of cancer and immunity required. Patients are monitored fortoxicities and other clinical responses.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may beadministered as part of the vaccine compositions. Such adjuvantsinclude, but are not limited to the following: polymers, co-polymerssuch as polyoxyethylene-polyoxypropylene copolymers, including blockco-polymers; polymer P1005; monotide ISA72; Freund's complete adjuvant(for animals); Freund's incomplete adjuvant; sorbitan monooleate;squalene; CRL-8300 adjuvant; alum; QS 21, muramyl dipeptide; trehalose;bacterial extracts, including mycobacterial extracts; detoxifiedendotoxins; membrane lipids; water-in-oil mixtures,water-in-oil-in-water mixtures or combinations thereof.

Suspending Fluids and Carriers

A variety of suspending fluids or carriers known to one of ordinaryskill in the art may be employed to suspend the vaccine compositions.Such fluids include without limitation: sterile water, saline, buffer,or complex fluids derived from growth medium or other biological fluids.Preservatives, stabilizers and antibiotics known to one of ordinaryskill in the art may be employed in the vaccine composition.

The following references are incorporated herein by reference in theirentirety: Jocham et al., Lancet 2004: 363, 594-599; Rosenberg, N. E.Journ. Med. 2004: 350, 14-1461-1463; Yamanaka et al., Brit. J. Cancer2003: 89, 1172-1179; Brossart, Transfusion & Apheresis Sci. 2002: 27,183-186; O'Rourke et al., Cancer Immunol. Immunother. 2003: 52, 387-395;Lotem et al., Brit. J. Cancer 2994: 90, 773-780; Hersey et al., CancerImmunol. Immunother. 2004: 53, 125-134; Limuna et al., J. Clin. Invest.2004: 113, 1307-1317.

The following experimental examples are illustrative in showing that adelipidation process of cancer cells occurred and in particular, thatthe cancer cell was modified and noted to exhibit a positive immunogenicresponse in the species from which it was derived. It will beappreciated that other embodiments and uses will be apparent to thoseskilled in the art and that the invention is not limited to thesespecific illustrative examples or preferred embodiments.

EXAMPLE 1 Delipidation Protocol

Delipidation of cancer cells can be achieved as follows. Cancer cellsshown in Table 2, for example at approximately 8×10⁵ cells in phosphatebuffered saline (PBS) were added to a final volume of 0.05% Triton X-100plus 3% diisopropylether (DIPE). The cancer cells were resuspended in 1ml saline. 0.05% Triton X-100 and DIPE was added to a finalconcentration of 3%. DIPE was added as 100% (30 μl of neat DIPE) to thecancer cell suspension and 5 μl of neat (100%) Triton was also added tomake a final volume of 1 mL cancer cell suspension. The solvent mixturecan also be made independently and then added to the cancer cellsuspension. The cancer cell suspension was mixed end-over-end at roomtemperature for 20 minutes. The sample was subsequently centrifuged at1000 rpm for 1 minute. Residual solvents were removed through a charcoalcolumn. Following the solvent removal step the cell suspension wasdiluted to a final volume of 2.5 ml. An aliquot of the resuspendedpellet was used to detect total cholesterol or total protein content toconfirm delipidation of the cancer cells, as described below.

It will be readily apparent to one of ordinary skill in the art, thatthe above procedure can be modified depending on the scale of thedelipidation. For example, in a large scale delipidation procedure orperhaps in a different solvent to cell suspension ratio, the bulksolvent layer can be removed and the residual solvents are eitherabsorbed through the use of charcoal or removed via centrifugation.

EXAMPLE 2 Total Cholesterol and Total Protein Content

An aliquot of the resuspension solution from Example 1 was used in atotal cholesterol assay. A further aliquot of the resuspension solutionfrom Example 1 was used in a total protein assay using commerciallyavailable kits. For example, 50 μl of the resuspension solution fromExample 1 was used in a Amplex Red Total Cholesterol Assay (MolecularProbes, Eugene, Oreg.). Another 50 μl of the resuspension solution wasused in a total protein assay using the BioRad Total Protein Assay(Bio-Rad Laboratories, Hercules, Calif.). The results from these studiesare presented in Table 3 which demonstrates that all delipidated cancercell lines, regardless of source, were successfully delipidated asobserved by the reduction in cholesterol concentration. Clearly, the 3%DIPE-0.05% triton X-100 protocol described in Example 1 efficientlyremoves lipids from a variety of cancer cells, including the murinecancer cell line B16-F10. Table 3 demonstrates >99% depletion ofcholesterol, while retaining >50% protein. Clearly, B16-F10 and TF-1cell lines were delipidated very efficiently. In addition, it appearsthe cell structure is maintained because of the good protein recoveryrates achieved.

TABLE 2 Cell Line Source Description TF-1 Human Erytholeukemia cell lineMT-2 Human CD4 T-cell line CEMx174 Human CD4 T-cell/B-cell line JAWS-IIMurine-C57BL/6 Immature dendritic cell line B16-F10 Murine-C57BL/6Melanoma cell line

TABLE 3 CHOLESTEROL PROTEIN CONCENTRATION CONCENTRATION CELL LINE(ug/ml) (ug/ml) undelipidated TF-1 125.3 78% protein recovery 8.36 97%cholesterol removal delipidated 98.3 0.28 undelipidated MT-2 108.8 33%protein recovery 4.09 >99% cholesterol removal  delipidated 36.1 0undelipidated CEMX174 100.8 34% protein recovery 4.54 >99% cholesterolremoval  delipidated 34 0 undelipidated JAWS-II 138.8 21% proteinrecovery 12.76 93% cholesterol removal delipidated 29.6 0.83undelipidated B16-F10 118.7 50% protein recovery 6.05 99% cholesterolremoval delipidated 68.7 0.06

EXAMPLE 3 Immature Dendritic Cell Uptake

Immature dendritic cells (DC) are classical antigen presenting cells(APC). These DCs efficiently take up whole pathogens, process cancercells/antigens, and present epitopes to B-cells for antibody production,and to T-cells for cell mediated immune responses. In this experiment,immature DCs were labeled with a dye (“A” in FIG. 1) that reads in thefluorescein isothiocyanate (FITC) channel of a fluorescence-activatedcell sorter (FACS) machine. Delipidated B16-F10 melanomas were labeledwith a separate dye (“C” in FIG. 1) that reads in the phycoerythrin (PE)channel of the FACS machine. Accordingly, if delipidated B16-F10 cellsare taken up by DCs, then fluorescence of the B16-F10 cells willdecrease, while DC fluorescence will remain intact.

The dendritic cell uptake protocol briefly comprised labelingdelipidated B16-F10 cells using the PKH26-GL (Red Dye Sigma-PE),incubated with immature JAWS-II cells labeled with PKH67-GL (Green DyeSigma-FITC), at a 1:1 ratio (1×10⁷:1×10⁷). After a 24 hour incubation,flow cytometric analysis was performed, and phagocytosis was defined bythe number of double-positive cells.

In order to stain at final concentrations of 2×10⁻⁶ M PKH26 dye and1×10⁷ cells/ml in a 2 ml volume, the following steps were performedusing aseptic techniques:

1. Adherent or bound cells were first removed using proteolytic enzymes(i.e., trypsin/EDTA) to form a single cell suspension.

2. All steps were performed at 25° C. A total of approximately 2×10⁷single cells were placed in a conical bottom polypropylene tube andwashed once using medium without serum.

3. The cells were centrifuged (400× g) for 5 minutes into a loosepellet.

4. After centrifugation, the supernatant was carefully aspirated leavingno more than 25 μl of supernatant on the pellet.

5. 1 ml of Diluent C (supplied with the staining kit) was added and thesolution (PKH67-GL (Green Fluorescent Cell Linking Dye, and PKH-GL (RedFluorescent Cell Linking Dye), Sigma, St. Louis, Mo.) resuspended bypipetting to insure complete dispersion.

6. Immediately prior to staining, 4×10⁻⁶ M PKH26 dye was prepared (as a2× stock) in polypropylene tubes using Diluent C To minimize ethanoleffects, the amount of dye added is less than 1% of the individualsample volume. If a greater dilution of the dye stock is necessary, anintermediate stock is made by diluting with 100% ethanol. Thepreparation remains at room temperature (25° C.).

7. 1 ml of cells (2×10⁷) was rapidly added to the 1 ml of PKH26 dye(1×10⁷ cells/ml). The sample was mixed by pipetting.

8. The solution was incubated at 25° C. for 2 to 5 minutes.Periodically, the tube was gently inverted to assure mixing during thisstaining period at 25° C.

9. To stop the staining reaction, an equal volume of serum or compatibleprotein solution (i.e., 1% BSA) was added and incubated for 1 min.

10. The serum-stopped sample was diluted with an equal volume ofcomplete medium.

11. The cells were centrifuged at 400× g for 10 minutes at 25° C. toremove cells from the staining solution.

12. The supernatant was removed and the cell pellet transferred to a newtube for further washing.

13. 10 ml of complete medium was added to wash the cells, that were thencentrifuged and resuspended to the desired concentration.

FIGS. 1 and 2 illustrate that DCs efficiently take up delipidatedB16-F10 cells.

EXAMPLE 4 In Vivo Studies—Therapeutic Vaccination

To test whether the efficient uptake of B16-F10 cells by DCs observed inExample 3 resulted in any therapeutic vaccination benefit, mice with B16cancers were vaccinated once with autologous DC pulsed with delipidatedB16-F10 cells.

Briefly, dendritic cell pulsing comprised pulsing 1×10⁶ immature bonemarrow derived dendritic cells (BMDDC) with 1×10⁶ delipidated B16-F10cells and 100 ng/ml Lipid A for 24 hours. The cells were incubated foran additional 24 hrs prior to adding Lipid A. The matured cells were fedwith GM-CSF and Lipid A by removing 2 ml of media and adding back 2 mlmedia of containing GM-CSF (10 ng/ml murine GM-CSF) and Lipid A.

Cancer antigen preparation required that the cancer cells to beutilized, for example B16-F10 cells, consisted of B16-F10 cells thatwere delipidated using the method of the instant invention, for examplea solvent mixture of 3% DIPE and 0.05% Triton X-100.

Mice were first challenged by injecting 2×10⁵ B16-F10 cells (50 μl) inPBS subcutaneously. In Vivo mice inoculations were then performed usingthe following test groups (5 mice per group): A. mice unimmunized (PBS);B. mice immunized with 2×10⁵ BMDDC only; C. mice immunized with 2×10⁵delipidated B16-F10 only; D. mice immunized with BMDDC pulsed withdelipidated B16-F10 cells. The experimental plan is shown in FIG. 11.

The experimental objective was to present a therapeutic cancer model.Many cancer patients have a pre-existing cancer, and one goal is tocontain cancer growth and therefore enhance the patients' survival.FIGS. 3 and 4 illustrate that, after just one boost of the vaccine, acontainment of cancer growth was observed in the vaccinated group,compared to the control group. In the experiment described above, thecontrol group was vaccinated with autologous DCs only. We believe thiscontrol group is more accurate than using a PBS control Group, sincevaccinated DCs could potentially take up autologous cancer cells andenhance an immune response against the cancer. An important feature ofthe experiment is that the results were observed after just onevaccination cycle. Many vaccination procedures require multiple roundsof vaccination to produce an immune response. Although vaccination withdelipidated B16-F10 cells reduced cancer growth compared to the PBSControl (FIG. 3), the reduction is not as drastic as when mice werevaccinated with DC pulsed with delipidated B16-F10 cells (FIG. 4). Theslight reduction could be due to the DCs present in the localized areaof vaccination taking up the delipidated B16-F10 cells and processingthem (as demonstrated in FIGS. 1 and 2). Overall, this experimentdemonstrated that vaccination with delipidated B16-F10 cells alone or asa dendritic cell hybrid protected against a pre-existing B16 melanomawith differing extents.

EXAMPLE 5 In Vitro Studies—Preventative Vaccination

To test whether DCs pulsed with delipidated B16-F10 cells had anybenefit in preventing mice from developing B16 cancers, mice werevaccinated once as described below and challenged with B16-F10 cancercells.

Briefly, cancer cells to be utilized, for example B16-F10 cellsconsisted of:

a) cells delipidated using the method of the instant invention, forexample a solvent mixture of 3% DIPE and 0.05% TRITON X-100; or

b) B16-F10 cells that were lysed using multiple freeze-thaw cycles.

In Vivo mice inoculations were then performed using the following testgroups (5 mice per group): A. mice unimmunized (PBS); B. mice immunizedwith 2×10⁵ BMDDC only; C. mice immunized with 2×10⁵ delipidated B16-F10cells only; D. mice immunized with BMDDC pulsed with delipidated B16-F10cells. The mice were challenged on day 6 after immunization by injecting5×10⁵ B16-F10 cells subcutaneously in the opposite flank. Theexperimental plan is shown in FIG. 12.

The experiments indicated that vaccination with DCs pulsed withdelipidated B16-F10 cells did not adequately protect mice from achallenge with B16-F10 cancer cells (FIGS. 5 and 6). However, we notedthat B16-F10 melanomas are considered a poor immunogenic tumor model,because they do not efficiently trigger an immune response. Therefore,we speculate that multiple rounds of vaccination may be effective in apreventative cancer model.

EXAMPLE 6 Therapeutic Vaccination and Cancer Antigen Specific ImmuneResponse

To determine whether vaccination with either delipidated B16-F10 cellsor DCs pulsed with delipidated B16-F10 cells generated a tumor antigenspecific immune response, antibody titers to B16 specific tumor antigensGP 100 and TRP-2 were measured.

An ELISA Assay was performed on Mouse Serum Samples as follows:

To coat the plates, peptides were diluted to 5 μg/ml in coating buffer(50 mM Tris, pH 9.5). Each well was coated with 100 μl/well on NuncImmulon HBX 96 well ELISA plates. Each plate was sealed and incubatedovernight. Protein was removed from the wells by flicking the plate andblotting on paper towels.

To block the plates, 200 μL blocking buffer (2% FBS, 1× PBS) was addedto all wells. Each plate was sealed and incubated 1-2 hours at 37° C.Each well was washed six times with 150 μL wash buffer (1× PBS, 0.05%Tween-20).

Primary Antibody was prepared as followed. Briefly, a 1:20 dilution ofmouse serum sample in sample dilution buffer (1× PBS, 5% Normal GoatSerum (NGS)) was prepared. Dilutions of 1:200, 1:400, 1:1000 and 1:2000were prepared. The plates were coated with 50 μL diluted serum andincubated at room temperature for 60 minutes. The plates were washed sixtimes with 150 μL wash buffer

Secondary Antibody was prepared as followed. Briefly, the secondaryantibody (Goat anti-mouse-HRP Fc Specific from Sigma) was diluted1:10,000 in sample dilution buffer. Each well was coated with 100μl/well and incubated at room temperature for 45 minutes. The wells werewashed six times with 150 μL wash buffer

ELISA development required adding 100 μL of TMB Substrate (Sigma) toeach well and incubated at room temperature for 5 minutes. The reactionwas stopped with 100 μL 1N H₂SO₄. The plates were read on a plate readerat absorbance 450 nm.

Vaccination with delipidated B16-F10 cells was observed to generatehigher antibody titers than the control group vaccinated with PBS (FIGS.7 and 8). However, the DC pulsed with delipidated B16-F10 cells had amuch higher overall antibody titer (FIGS. 9 and 10), indicating that theDC pulsed with delipidated B16-F10 cells substantially enhanced antigenprocessing and presentation. Antibody titers in animals receiving DCspulsed with delipidated B16-F10 cells were much higher than titers inanimals vaccinated with DCs alone. The results demonstrate thatdelipidated B16-F10 cells are efficiently processed and presented byDCs, which leads to an enhanced immune response. Vaccination with DCalone was also observed to enhance antibody responses (FIGS. 9 and 10),possibly due to the incoming DCs taking up already existing cancer cellsand processing them for an immune response. However, the uptake,processing and/or presentation rates were lower than animals receivingDC pulsed with delipidated B16-F10 cells. Overall, we observed thatdelipidated B16-F10 cell vaccination enhanced antibodies to B16 tumorantigens TRP-2 and GP 100 (FIGS. 7 and 8). Furthermore, we noted thatanimals receiving DCs pulsed with delipidated B16-F10 cell vaccinationdisplayed greatly enhanced antibody titers to B16 tumor antigens TRP-2and GP 100 (FIGS. 9 and 10). Although we observed that mature DCs cantake up already existing cancers from mice, process them and presentthem to the immune system, this process is not as efficient as when DCsare pulsed with the delipidated B16-F10 cells.

EXAMPLE 7 In Vivo Experimental Protocol for Harvest and Growth of BoneMarrow Derived Dendritic Cells

The following describes a method for the in vivo harvesting and growthof bone marrow derived dendritic cells (BMDDC). In this experiment themouse species used was C57BL/6, approximately 8-16 weeks old. Five micewere assigned to each group. The cancer model in this experiment was aB16-F10 melanoma (in C57BL/6 mice), known to be a poorly immunogenictumor model. Bone marrow was harvested from femurs and tibiae through aFalcon 100-μm nylon cell strainer.

Bone Marrow Preparation: Femurs and tibiae of female, 4-12 week oldC57BL/6 mice were removed and purified from the surrounding muscletissue by rubbing with Kleenex tissues. Intact bones were left in 70%ethanol for 2-5 minutes for disinfection and washed with PBS. Both endsof the bones were cut with scissors and the marrow flushed with PBSusing a Syringe with a 0.45 mm diameter needle. Vigorous pipetting wasused to disintegrate clusters within the marrow suspension. Thesuspension was then washed once with PBS. The cells were resuspended in(R10) RPMI-1640 (GIBCO BRL) supplemented with Penicillin (100 U/mL,Sigma), Streptomycin (100 U/mL, Sigma), L-glutamine (2 mM, Sigma),2-mercaptoethanol (50 uM, Sigma), 10% heat-inactivated and filteredFetal Calf Serum all filtered through a (0.22 uM, Millipore or CorningFilter)

Day 0: Seed Bone Marrow. Leukocytes were seeded at 2×10⁶ per 100 mm dishin 10 ml R10 medium containing 200 U/ml (=20 ng/ml) rmGM-CSF.

Day 3: Another 10 ml of R10 medium containing 200 U/ml rmGM-CSF wasadded.

Day 6: Half of the culture supernatant was collected, the removedculture supernatant was centrifuged, and the pellet was resuspended in10 ml of fresh R10 medium containing 200 U/ml rmGM-CSF. The suspensionwas re-plated back onto the original plate.

Day 7: The immature DCS were fed with lysed or delipidated B16-F10cells. Add 1×10⁶ DC and 1×10⁶ delipidated B16-F10 cells for 24 hours.

Complete Maturation:

Day 8: The DC and delipidated B16-F10 cells were matured using Lipid Aat 100 ng/ml or lipopolysaccharide (LPS) at 1 ug/ml+30-100 U/ml rm(recombinant murine) GM-CSF. The cells were incubated for an additional24 hrs prior to injection.

Cell yield evaluation: Cultured cells were washed once. An aliquot ofcells was mixed 1:1 (vol:vol) with trypan blue Solution (Sigma). Trypanblue negative, large leukocytes (erythrocytes excluded by size andshape) were counted as viable under the microscope.

FACS Analysis

1×10⁵ cells were stained with 50 μl hybridoma culture supernatantscontaining 0.1% sodium azide or purified first and second stepantibodies see M. B. Lutz et al. Journal of Immunological Methods223(1999)77-92 79. The cells and primary and secondary antibody (5-20μg/ml) were incubated for 30 min on ice. Both primary and secondaryreagents were diluted in PBS containing 5% fetal calf serum (FCS) and0.1% sodium azide, which also served as washing medium. Samples wereanalyzed with a FACScan (Becton Dickinson, Heidelberg, Germany).

The following antibodies were used for surface and cytoplasmic stainingas culture supernatants reviewed in Leenen et al., 1997: MHC molecules:H-2 K-FITC_M1r42, rat IgG2a.; (Pharmingen, San Diego, Calif.);co-stimulatory adhesion molecules: CD80_B7-1-PE, 16-10A1, hamster IgG,(Pharmingen), CD86_B7-2-FITC, GL1, rat IgG2a, (Pharmingen);CD40-PE-HM40-3, rat IgG2b, (Pharmingen). DC markers: CD25_IL-2Ra, 7D4,rat IgM., NLDC-145 DEC-205, rat IgG2a., CD11c PE_HL3, hamster IgG-PE.,G235-2356 (Pharmingen).

Endocytotic capacity of BMDDC was investigated as described in detailelsewhere, see Sallusto et al., 1995 and Lutz et al., 1997. Briefly,2×10⁵ cells were incubated with FITC-DX at 1 mg/ml on ice surfacebinding but no endocytosis or in a 37° C. surface binding andendocytosis waterbath for 30 min. Cells were washed with ice cold PBSand stained for surface MHC class II molecules as described above.

Whole bone marrow cells were plated in six-well plates in complete IMDM(2 mM glutamax, 100 U/ml penicillin, 100 ug/ml streptomycin, 50 uM 2-ME,and 5% FCS) supplemented with 10 ng/ml murine GM-CSF, and 20 ng/mlmurine IL-4. Cultures were fed every 2-3 days by removing 50% of mediumfrom each well and adding back an equal amount of fresh growth factorsupplemented cIMDM. The cultures were maintained for 6-8 days. Nonadherent and loosely adherent cells were harvested, washed and used forin vitro and in vivo experiments.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It will be appreciated that otherembodiments and uses will be apparent to those skilled in the art andthat the invention is not limited to these specific illustrativeexamples or preferred embodiments. It should be understood, of course,that the foregoing relates only to preferred embodiments of the presentinvention and that numerous modifications or alterations may be madetherein without departing from the spirit and the scope of the inventionas set forth in the appended claims.

1-23. (canceled)
 24. A method of treating or delaying progression of acancer in an animal or a human comprising: administering to the animalor the human a composition comprising a modified cancer cell withreduced lipid content, as compared to an unmodified cancer cell of thesame cancer type as the modified cancer cell, and at least one cancercell associated antigen, wherein the unmodified cancer cell is of thesame cancer type as the cancer in the animal or the human, and apharmaceutically acceptable carrier, wherein the composition isadministered in an amount effective to treat or delay the progression ofthe cancer in the animal or the human.
 25. The method of claim 24,wherein the composition further comprises an immunostimulant, adendritic cell or a combination thereof.
 25. The method of claim 24,wherein the modified cancer cell is produced by a process comprising:contacting the unmodified cancer cell with a first extraction solvent;mixing the fluid and the first extraction solvent for a sufficient timeto extract lipid from the unmodified cancer cell, thereby producing themodified cancer cell; separating the first extraction solvent from thefluid; and, collecting the fluid containing the modified cancer cell.